European Journal of Pharmacology 775 (2016) 1–14

Contents lists available at ScienceDirect

European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Review

Cannabinoid pharmacology in cancer research: A new hope

for cancer patients?
Farideh A. Javid a,n, Roger M. Phillips a, S. Afshinjavid b, Roberta Verde c, Alessia Ligresti c
a
Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, UK
b
Faculty of Science and Engineering, University of Chester, Pool lane, Chester CH2 4NU, UK
c
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, National Research Council of Italy, Pozzuoli, NA, Italy

art ic l e i nf o a b s t r a c t

Article history: Cannabinoids have been used for many centuries to ease pain and in the past decade, the endocannabinoid
Received 4 June 2015 system has been implicated in a number of pathophysiological conditions, such as mood and anxiety dis-
Received in revised form orders, movement disorders such as Parkinson's and Huntington's disease, neuropathic pain, multiple
5 January 2016
sclerosis, spinal cord injury, atherosclerosis, myocardial infarction, stroke, hypertension, glaucoma, obesity,
Accepted 3 February 2016
and osteoporosis. Several studies have demonstrated that cannabinoids also have anti-cancer activity and as
Available online 5 February 2016
cannabinoids are usually well tolerated and do not produce the typical toxic effects of conventional che-
Keywords: motherapies, there is considerable merit in the development of cannabinoids as potential anticancer
Cannabinoids therapies. Whilst the presence of psychoactive effects of cannabinoids could prevent any progress in this
Cancer field, recent studies have shown the value of the non-psychoactive components of cannabinoids in activating
Cannabinoid receptors
apoptotic pathways, inducing anti-proliferative and anti-angiogenic effects. The aforementioned effects are
suggested to be through pathways such as ERK, Akt, mitogen-activated protein kinase (MAPK) pathways,
phosphoinositide 3-kinase (PI3K) pathways and hypoxia inducible factor 1 (HIF1), all of which are important
contributors to the hallmarks of cancer. Many important questions still remain unanswered or are poorly
addressed thus necessitating further research at basic pre-clinical and clinical levels. In this review, we ad-
dress these issues with a view to identifying the key challenges that future research needs to address.
& 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Anti-tumour effects of cannabinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Abbreviations: ABCC1, ATP-binding cassette (ABC) transporter; AC, adenylyl cyclase; AEA, anandamide; AKt, protein kinase B; AM251, a CB1 receptor antagonist; AM630, a
CB2 receptor antagonist; AMPK, 5′-adenosine monophosphate-activated protein kinase; ATF-4, activating transcription factor-4; AR, androgen receptors; CBD, cannabidiol;
CB1IR, CB1 receptor immunoreactivity; Cdk, cyclin-dependent kinase; Chk 1, cell cycle checkpoint; COX2, cyclooxygenase-2; CXCR4, chemokine receptor 4; CXCL12, a
chemokine protein encoded by the CXCL12 gene; Δ9-THC, Δ9-tetrahydrocannabinol; EGFR, epidermal growth factor receptor; ER, estrogen; ERK, extracellular signal-
regulated kinase; FAAH, fatty acid amide hydrolase; FAK, focal adhesion kinase; GBM, glioblastoma multiform; Gi/o, a subunit of G protein; GTPγS, guanosine 5′-O-[gamma-
thio] triphosphate; HER2, human epidermal growth factor receptor 2; HIF-α, hypoxia-inducible factor; HU-210, highly potent cannabinoid receptor agonist, 96aR0-trans-3–
91, 1-Dimethylheptyl)-6a, 7, 10, 10a-tetrahydro-1-hydroxy-6, 6-dimethyl-6H-dibenzo [b,d]pyran-9-methanol; ICAM, intracellular adhesion molecule; JWH-015, a selective
CB2 agonist, 2-Methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone; JWH-133, a potent selective CB2 agonist, (6aR, 10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-Tetra-
hydro-6,6,9-trimethyl-6H-dibenzo [b,d]pyran MAPK, mitogen activated protein kinase; LAK, lymphokine-activated killer; LPI, lysophosphatidylinositol; MAGL, monoglycerol
lipase; MMP-2, matrix metallopeptidase 2; MMP-9, matrix metallopeptidase 9; MRP1, Multidrug resistance-related protein 1; mTOR, mammalian target of rapamycin; NF-
κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NGF, nerve growth factor; PD98059, p42/44 inhibitor, 2-(2-Amino-3-methoxyphenyl)-4/h-1-benzopyran-
4-one; PGE2, prostaglandine E-2; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PCNA, proliferating cell nuclear antigen; PPARs, peroxisome proliferator-activated
receptors; PR, progesterone; PRLr, prolactin receptor; PSA, prostate specific antigen; PyMT, polyoma middle T oncoprotein; RAF-1, a proto-oncogene, serine/threonine
kinase; ROS, reactive oxygen species; RXRα, retinoid X receptor; SB203580, a p38/MAPK inhibitor, 4-[5-(4-Fluorophenyl)-2-[4-(methylsulphonyl)phenyl]-1H-imidazol-4-yl]
pyridine; SC58236, COX-2-specific inhibitor, 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide; siRNA, small interfering RNA; SR141716, Ri-
monabant, a selective CB1 receptor antagonist or an inverse agonist; Src gene, a family of proto-oncogenic tyrosine kinases; Th1, a type of T helper cells; Th2, a type of T
helper cells; TIMP-1, tissue inhibitor of matrix metalloproteinases-1; TrK A, tropomyosin receptor kinase A; TRPM8, transient receptor potential channels of melastatin-type
8; TRPVA1, transient receptor potential A1; TRPV1, transient receptor potential vanilloid 1; TRB3, tribbles homologue that inhibits Akt/PKB activation; 2-AG, 2-arachidonoyl
glycerol; VEGF, vascular endothelial growth factor; WIN 55,212–2, a CB1 and CB2 receptor agonist [(R0-( þ )-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-
de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanoneesylate]
n
Correspondence to: Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK.
E-mail address: fajavid@hud.ac.uk (F.A. Javid).

http://dx.doi.org/10.1016/j.ejphar.2016.02.010
0014-2999/& 2016 Elsevier B.V. All rights reserved.
2 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14

3. Cannabinoids and breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

4. Cannabinoids and brain cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5. Cannabinoid and lung cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6. Cannabinoids and intestinal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
7. Cannabinoids and reproductive system cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
8. Conclusion and future direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Conflict of interests/acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1. Introduction liver disorder, pain and atherosclerosis (Buckley, 2008).

Both CB1 and CB2 receptors are metabotropic and belong to the
It is known that cannabinoids, the active components of Can- G-protein coupled receptor family (Howlett et al., 2002). Activa-
nabis sativa, act by mimicking the endogenous substances (the tion of CB1 and CB2 receptors stimulates cellular signalling via
endocannabinoids anandamide and 2-arachidonoylglycerol (2- alpha subunit of G protein (Gi/o), leading to inhibition of adenylate
AG)) by activating specific cell-surface cannabinoid receptors cyclase and the subsequent activation of many other pathways
(Devane et al., 1992). Currently, the cannabinoid receptor ligands such as mitogen-activated protein kinase (MAPK) pathways,
are generally divided into three main categories known as phy- phosphoinositide 3-kinase (PI3K) pathways, modulation of ion
tocannabinoids, endogenous cannabinoids and synthetic canna- channels (through CB1 receptors), protein kinase B (Akt), ceramide
binoids (Fig. 1). After the clarification of the chemical structure of signalling pathways in tumour cells and modulation of cycloox-
(-)-Δ9-tetrahydrocannabinol (Δ9-THC) which is the primary psy- ygenase-2 (COX-2) signalling pathway (Demuth and Molleman,
choactive component of the cannabis plant (Gaonia and Mechou- 2006; Galve-Roperh et al., 2000; Glass and Northup, 1999; Guz-
lam, 1964a, 1964b), other chemically related terpenophenolic man et al., 2001; Qamri et al., 2009).
compounds were identified in Cannabis sativa, including canna- There is also pharmacological evidence that non-CB1 and non-CB2
bichromene (CBC) (Gaoni and Mechoulam, 1966) and cannabigerol receptors mediate the actions of cannabinoids located in the brain
(CBG) (Gaoni and Mechoulam, 1964c). Although the pharmacology (Breivogel et al., 2001; Di Marzo et al., 2000). The hypothesis that
of most of the cannabinoids is unknown, Δ9-THC is the most putative CB3 or non-CB1/CB2 receptor exist is supported by the fact that
widely studied owing to its high potency and abundance in can- some of the anandamide (AEA)-mediated effects were neither in-
nabis (Pertwee et al., 2010). Among the herbal cannabinoids, other hibited by selective CB antagonists nor fully abolished in knockout mice
relevant plant-derived cannabinoids include Δ8-THC, which is al- lacking CB1 receptors (De Petrocellis and Di Marzo, 2010). Recent ad-
most as active as Δ9-THC but less abundant and cannabinol (CBN), vances suggest, at least for AEA, that the transient receptor potential
which is produced in large amounts but is a weak cannabomimetic vanilloid 1 receptor (TRPV1) channel may be considered as the “third”
agent. Cannabidiol (CBD), CBG and CBC are devoid of psychoactive receptor involved in endocannabinoid signalling (Di Marzo et al., 2001;
potential. The chemical structures of some cannabinoids are Ross, 2003). For example, it has been shown that the endocannabinoids
shown in Fig. 1. exert their apoptotic effect by binding to TRPV1, a non-selective cation
So far, two cannabinoid-specific receptors CB1 and CB2 have channel targeted by capsaicin, the active component of hot chilli pep-
been cloned and characterized from mammalian tissues (Howlett pers (Smart and Jerman, 2000). However, the precise role of this re-
et al., 2002). Mouse CB1 receptor and CB2 receptor share 66% ceptor in cannabinoid signalling is still unclear and this uncertainty
overall homology and 78% in the transmembrane region (Shire extends into the cancer field where its potential role in cancer biology
et al., 1996). Human CB1 receptor and CB2 receptor share an overall (proliferation and migration of cancer cells) and cancer pharmacology
homology of 44%, and 68% in the transmembrane region respec- (resistance to chemotherapeutic agents) needs further investigation
tively (Munro et al., 1993). Homology (96%) has been reported (Lehen'kyi and Prevarskaya, 2011; Liberati et al., 2013). Evidence also
between human and mouse CB1 receptor (Chakrabarti et al., 1995), exists supporting a role for peroxisome proliferator-activated receptors
whilst human and mouse CB2 receptors share 82% homology (PPARs) in the actions of cannabinoids (Sun and Bennett, 2007). More
(Shire et al., 1996). Many central and peripheral effects have been recent studies have provided evidence for the interaction of cannabi-
associated with the activation of CB1-receptors (Matsuda et al., noids with the orphan receptors such as G protein receptor 55 (GPR55)
1990; Munro et al., 1993; Pertwee, 2006; Pertwee et al., 2010). The (Andradas et al., 2011; Pineiro et al., 2011). Thus in addition to CB1 and
CB2 receptor, originally thought of as being exclusively present in CB2 receptors other targets might be involved in mediating an effect to
the immune system, is highly expressed in B and T lymphocytes, cannabinoids and endocannabinoids.
macrophages and in tissues such as the spleen, tonsils and lymph The potential of cannabinoids to alleviate pain has been re-
nodes (Herkenham et al., 1991; Howlett et al., 2002; Porter and cognised for many centuries. The antinociceptive actions are
Felder, 2001; Pertwee et al., 2010). Recently CB2 receptors have mediated via both the CB1 and CB2 receptors (Pacher et al., 2006).
been shown to be also located in the brain stem (Van Sickle et al., This does not negate a role for other receptors such as TRPV1,
2005). Further studies using CB1 knockout mice demonstrated that transient receptor potential cation channel A1(TRPA1), orphan
CB1 receptors are involved in a variety of different behavioural GPCR (i.e. GPR55) or PPAR-γ (Maione et al., 2006, 2013; Perez-
disorders such as depression, anxiety, feeding and cognition as Gomez et al., 2013; Moreno et al., 2014). For a long time, the de-
well as pain at the peripheral, spinal and supraspinal levels (Val- velopment of cannabinoids as anticancer agents has been re-
verde et al., 2005). Such studies using CB1 knockout mice also stricted to two therapeutic avenues (antiemetic and analgesic).
revealed the interactions between different systems such as They have therefore been evaluated in terms of palliative care as
opioids, gamma aminobutyric acid (GABA) and cholecystokinin cannabinoids can play an important role in the relief of pain,
(CCK) via CB1 receptors (Valverde et al., 2005). CB2 knockout mice nausea, vomiting, and stimulation of appetite in cancer patients.
have also been developed and revealed/confirmed the involve- However, the involvement of CB receptors in pain and their use in
ment of CB2 receptors in a variety of different systems such as the palliative care in cancer patients are not the focus of this re-
immune system, inflammation, apoptosis, chemotaxis, bone loss, view. In the present review, the aim is to focus on the anti-tumour
 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14 3

Phytocann
nabinoids:

Cannaabidiol (CBD
D) Delta-9- Tetrahydrocaannabinol (Δ9-THC)

Synthetic cannabinoids:

HU 210 WIN 552212-2

Endocann
nabinoids:

Ananndamide (A
AEA) 2-AG
Fig. 1. Chemical structures of some cannabinoids.

effects of cannabinoids, identify potential mechanisms by which
cannabinoids induce anti-tumour effects and discuss the potential
and challenges for the future development of this class of
compound.
2. Anti-tumour effects of cannabinoids
Whilst cannabinoids exert palliative effects in cancer patients
by preventing nausea, vomiting and pain and by stimulating ap-
petite, they have also been shown to inhibit the growth of tumour
cells in culture and animal models by modulating key cell-sig-
nalling pathways. In 1975, Munson and collaborators were the first
to report the anti-proliferative properties of cannabis compounds
(Munson et al., 1975). They showed that Δ9-THC inhibits lung-
adenocarcinoma cell growth in vitro and after oral administration
in mice. It was not until the late 1990s however that further stu-
dies on the anti-cancer effects of cannabinoids were carried out.
Several plant-derived (for example, Δ9-THC and CBD), synthetic
(for example, WIN-55, 212–2 and HU-210) and endogenous can-
nabinoids (for example, AEA and 2-AG) are now known to exert
anti-proliferative actions on a wide range of tumour cells in vitro
(Guzman et al., 2002). The involvement of CB1 and/or CB2 re-
ceptors in the anti-tumour effects of cannabinoids has been shown
by various biochemical and pharmacological approaches, in par-
ticular by determining cannabinoid-receptor expression and by
4 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14
using selective cannabinoid-receptor agonists and antagonists
(Guzmán, 2003; Sarfaraz et al., 2008; Pisanti et al., 2013 ;Velasco
et al., 2012). Such studies have shown that cannabinoids can
prevent proliferation, metastasis, angiogenesis and exert pro-
apoptotic effects in a variety of cancer cell types such as lung,
breast, prostate, skin, intestine, glioma, lymphoma, pancreas and
uterus (Blazquez et al., 2006; Carracedo et al., 2006b; Casanova
et al., 2003; Cianchi et al., 2008; Galve-Roperh et al., 2000; Guz-
mán, 2003; Pacher et al., 2006; Sánchez et al., 2001a, 2001b). Si-
lencing CB2 receptors with specific small interfering RNA (siRNA)
in prostate cancer cells (PC-3) revealed the involvement of CB2
receptors in the growth inhibition of prostate cancer cells (Olea-
Herrero et al., 2009) via stimulation of autophagy. Cannabinoid-
induced AMPK activation of autophagy was also confirmed in vivo
(Vara et al., 2011). The over expression of cannabinoid receptors
and elevated endocannabinoid levels have also been reported in a
variety of different cancer types such as prostate, skin, hepato-
cellular carcinoma, colon, endometrial sarcoma, glioblastoma
multiforme (GBM), meningioma and pituitary adenoma (see Ta-
ble 1) (Blazquez et al., 2006; Xu et al., 2006; Pisanti et al., 2013).
CB1 receptors are also up-regulated in Hodgkin lymphoma cells
and also in chemically induced hepatocarcinoma (Benz et al., 2013;
Mukhopadhyay et al., 2015). Also the expression of CB1 and CB2
receptors was found to increase in mantel cell lymphoma and fatty
acid amide hydrolase (FAAH) expression was reduced when
compared to non-malignant B-cells (Islam et al., 2003; Ek et al.,
2002; Wasik et al., 2014). It has also been shown in both the
mouse model of metastatic melanoma and in humans that the
circulating endocannabinoid levels have been associated with an
increase in disease progression (Sailler et al., 2014). Indeed many
reports have shown that an increase in the level of en-
docannabinoids and their receptors correlates with tumour ag-
gressiveness (Malfitano et al., 2011) and that cannabinoids can
inhibit the growth of xenograft tumours (Blazquez et al., 2006;
Carracedo et al., 2006b; Sánchez et al., 2001a).
In neoplastic cells cannabinoids have also been shown to in-
hibit angiogenesis and directly initiate apoptosis or cell cycle ar-
rest (Blazquez et al., 2006; Carracedo et al., 2006b; Sánchez et al.,
2001a). Other studies have shown the ability of cannabinoids to
affect cellular signalling pathway/s essential for cell survival and
growth (Bifulco et al., 2008; Kogan, 2005). Some studies have also
demonstrated and suggested the involvement of autophagy in the
mechanism of cannabinoid-induced cytotoxicity (Armstrong et al.
2015; Vara et al., 2011; McAllister et al., 2015). Cannabinoids have
also been reported to inhibit nerve growth factor (NGF)-induced
Table 1
Tumours, which are sensitive to cannabinoid-induced growth inhibition.
Tumour type Experimental system
Lung carcinoma In vivo (mouse)
In vitro (isolated lewis lung cells)
Glioma In vivo (mouse, rat)
In vitro (C6 glioma cells)
Thyroid epithelioma In vivo (mouse)
In vitro (KiMol cells)
Lymphoma/leukaemia In vivo (mouse)
In vitro (MCL cells; Jurkat cells; Molt-4 cells;
Sup-T1 cells)
Skin carcinoma In vivo (mouse)
In vitro (PDV.C57 cells; HaCa4 cells)
Uterus carcinoma In vitro (HeLa cells)
Breast carcinoma In vitro (MCF-7 cells)
Prostate carcinoma In vitro (LNCaP cells; DU-145 cells; PC3 cells)
Neuroblastoma In vitro (CHP100 cells)
N.D., not determined; TRPV1, type 1 vanilloid receptor.
proliferation of PC-3 cells (Melck et al., 2000) through interaction
with CB1 receptors and synthetic endocannabinoid-vanilloid hy-
brids via stimulation of TRPV1 channels. However, other studies
demonstrate that cannabinoid-induced anti-tumour activity was
only marginally dependent upon the interaction between canna-
binoid and TRPV1 receptors (Massi et al., 2004; Torres et al., 2011;
Vaccani et al., 2005). On the other hand, cannabinoids have also
been shown to actively induce apoptosis in these cells via a CB1
receptor-independent mechanism as the Δ9-THC-induced apop-
tosis was not reversed by the CB1 receptor antagonist, SR141716
(Ruiz et al., 1999).
The effects of cannabinoids in modulating cell cycle and sig-
nalling pathways are diverse (Fig. 2) and may depend on the type
of tumour cells. Whilst there is evidence showing that some
cannabinoids induce anti-proliferative effect on tumour cells in
vitro (Hart et al., 2004; White et al., 1976; Velasco et al., 2015),
there is evidence to suggest that the effects may depend upon the
disease context with differential effects seen in different tumour
types. The subsequent sections focus specifically on the evidence
of cannabinoid induced anti-tumour effects in specific cancer
types with the aim of exploring the mechanism of action of non-
psychoactive components of cannabinoids in a disease specific
context.
3. Cannabinoids and breast cancer
The first report on the antineoplastic property of cannabinoids
in breast cancer are in the late 1990s, when it was shown that pre-
treatment with the endocannabinoid anandamide inhibited pro-
lactine- and nerve growth factor-induced proliferation of two
hormone-sensitive, estrogen and progesterone (ER þ /PR þ ) breast
cancer cell lines (EFM-19 and MCF-7 cell lines). In this case,
treatment reduced the levels of prolactin receptor (PRLr) and
nerve growth factor receptors via CB1 receptor activation. Thus,
the inhibition of adenylyl cyclase activity that in turn induced
prolonged activation/stimulation of the RAF1-MEK-ERK cascade,
leads to a down-regulation of the PRL receptor and levels of TrkA
NGF receptors (De Petrocellis et al., 1998; Melck et al., 2000, 1999).
Of interest, other studies showed that in both MCF-7 cells and
tamoxifen-resistant MCF-7 (TAMR-MCF-7) tumour cells, anti-an-
giogenesis effects exerted by novel synthetic hexahydrocannabinol
analogueues was through the suppression of VEGF (Thapa et al.,
2011). It should be noted that both MCF-7 and TAMR-MCF-7 cells
have shown a strong association between enhanced VEGF
Effect Receptor Ref.
Decreased tumour size N.D. Munson et al. (1975)
Cell-growth
inhibition
Decreased tumour size CB1, CB2 Galve-Roperh et al. (2000), Sánchez et al. (2001a) and Ja-
Apoptosis cobsson et al. (2000)
Decreased tumour size CB1 Bifulco et al. (2001)
Cell-cycle arrest
Decreased tumour size CB1/CB2 McKallip et al. (2002) and Gustafsson et al. (2006)
Apoptosis
Decreased tumour size CB1, CB2 Casanova et al. (2003)
Apoptosis
Cell-growth inhibition N.D. Mon et al. (1978), Blevins and Smith (1980)
Cell-cycle arrest CB1 DE Petrocellis et al. (1998), Melck et al. (2000)
Apoptosis CB1/CB2 Mimeault et al. (2003), Melck et al. (2000), Ruiz et al. (1999),
Sarfaraz et al. (2005)
Apoptosis Trpv1 Maccarrone et al. (2000)
 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14 5

Activation of cannabinoids receptors

N N

C C
c-AMP EGFR Activation of Ceramide

PKA phosphorylation of ERK1/2 , JNK1/2 , Akt, FAK ERK1/2 PI3K and Akt Bad protein p38 p8

FAK tyrosine phosphorylation Cyclins ATF-4, CHOP and TRB3

MAPK activation

Inactivation of tyrosine phosphatases Cdks caspases

Anti-proliferative Migration
effects Migration,
invasion & growth

Apoptosis

Fig. 2. Schematic representation showing some examples of pathways activated following the activation of cannabinoid receptors.

Agonists/endocannabinoids binding at cannabinoid receptors

N N

C C
cAMP erbB Ras loss in Cdk2 activity Cox-2 Chk1 activation and
Cdc25A proteolysis

PKA RAF1 p2/waf PGE2 dephosphorylation

through RAF1/MEK/ERK cyclin E/Cdk2 kinase of Thr14/Tyr15
cascade activation

MAPK PRL and trk level prevention of Cdk2 activation

activation

Anti-proliferative Angiogenesis Cell arrest in S phase of

effect & apoptosis the cell cycle
Anti-proliferative
effect at the S
phase

Fig. 3. Schematic representation of examples of different pathways associated with anti-proliferative effects induced by cannabinoid receptor activation in breast cancer.

production and more aggressive phenotype (Kim et al., 2008,
2009). The anti-angiogenesis activity afforded by the novel syn-
thetic cannabinoids were shown to be independent of CB1 and CB2
receptor activity and through an inhibition of NF-κB transcrip-
tional activity which in turn plays an important role in VEGF
regulation and angiogenesis (Thapa et al., 2011). Further studies
demonstrated that the proliferation of EVSA-T, a hormone-sensi-
tive (ER  /PR þ ) breast cancer cell line, was also inhibited by TCH
via CB2 receptor activation. This caused the activation of the
transcription factor JunD, the up-regulation of gene expression
and subsequent translocation of protein to the nuclear compart-
ment (Caffarel et al., 2008). Thus, it seems that cannabinoids do
activate many cell-specific pathways, however not all pathways
are simultaneously stimulated (Fig. 3). In addition to their anti-
mitogenic property, cannabinoids have been also shown to play a
role in hormone-sensitive breast cancer cell migration and inva-
sion. Specifically, CB2 has recently been found to modulate breast
tumour growth and metastasis by inhibiting signalling of the
chemokine receptor CXCR4 and its ligand CXCL12 in both in MCF7
overexpressing CXCR4 and NT2.5 injected immune-competent
syngenic FVB mice (Nasser et al., 2011). However, other studies
showed that Δ9-THC enhanced breast cancer growth and metas-
tasis although specifically in cells expressing low levels of canna-
binoid receptors (i.e. mouse mammary carcinoma 4T1 cells) by
suppressing the antitumour immune response (McKallip et al.,
2005). Such studies showed that exposure to Δ9-THC led to an
increase in the level of cytokines such as IL-4 and IL-10, suggesting
that Δ9-THC exposure may specifically suppress the cell-mediated
Th1 response by enhancing Th2-associated cytokines (McKallip
et al., 2005). Among the hormone-sensitive histopathological
6 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14
subtypes, it should be mentioned that in breast tumours that ex-
press the tyrosine kinase receptor HER2, cannabinoids have been
shown to be very effective. In fact, in addition to the correlation
between tumour aggressiveness and CB2 receptor expression in
breast cancer, a significant correlation between CB2 receptor and
ErbB2 has been recently demonstrated (Caffarel et al., 2010). In
particular, this study showed that the selective agonist JWH-133
was as effective as Δ9-THC (a CB1/CB2-mixed agonist) in reducing
tumour growth and progression through inhibition of the pro-
tumourigenic kinase Akt pathway. Moreover, in vivo studies re-
ported that Δ9-THC was efficacious in reducing tumour growth,
tumour number, and the amount/severity of lung metastases in
MMTV-neu mice (Caffarel et al., 2010).
Another group of clinically important breast tumours are triple-
negative tumours. These tumours are characterized by the total
lack of expression in ER, PR or HER2. Numerous pieces of evidence
both in vitro and in vivo, demonstrate that cannabinoids (acting
through a plethora of different mechanisms) can be considered as
promising candidates for the treatment of ER  /PR  /HER2  breast
cancer. The metabolically stable anandamide analogueue (Met-F-
AEA), significantly affected adhesion and migration of both the
highly invasive human breast carcinoma cell line (MDA-MB-231)
and murine breast cancer cell line (TSA-E1) by reducing FAK tyr-
osine phosphorylation/activation and Src phosphorylation via CB1
receptor (Grimaldi et al., 2006). Interestingly, the same group re-
ported that pre-treatment with SR141716 could also inhibit tu-
mour growth or induce apoptosis possibly through an inhibition of
ERK1/2 signalling in lipid rafts and caveolae (Sarnataro et al.,
2006). Both are highly implicated in tumour growth and metas-
tasis in breast cancer (Sloan et al., 2004; Williams et al., 2004).
Later studies reported an over- expression of cannabinoid re-
ceptors in primary human breast tumours compared with normal
breast tissues, as well as in breast cancer cell lines MDA–MB231
and MDA–MB468 (Qamri et al., 2009). Such studies have shown
that stimulation of both CB1 and CB2 receptor by their agonists,
WIN-55,212–2 and JWH-133, respectively inhibited cell prolifera-
tion and migration in breast cell lines. The results were in line with
in vivo findings where in mammary gland tumours in the polyoma
middle T onco-protein (PyMT) transgenic mice model, a geneti-
cally engineered model of triple negative breast cancer, WIN-
55,212–2 or JWH-133 showed a reduction in tumour growth and
lung metastasis. Inhibition induced by the agonists was sensitive
to antagonism by CB1 and CB2 antagonists AM 251 and SR144528,
suggesting involvement of CB1 and CB2 receptors.
In terms of signal transduction pathways, cyclooxygenase-2
and prostaglandin E2, via the regulation of GTPases and tran-
scription factors, have been implicated in the action of cannabi-
noids on breast cancer growth and metastasis (Qamri et al., 2009).
Such studies also reported a significant reduction in angiogenesis
(CD31 staining) and attenuation of proliferation (Ki67 staining). A
reduction in the level of Cdc42 activity and nuclear expression of
transcription factors c-Jun and c-Fos in MDA–MB231 cells were
also reported (Qamri et al., 2009).
The involvement of CB1 as well as TRPV1 receptors on the in-
vasiveness of MDA-MB-231 cells has been recently discussed. Se-
lective agonists were shown to reduce cell invasion and accord-
ingly MMP-2 expression (Farsandaj et al., 2012). In addition, a
down-regulation of vascular endothelial growth factor and con-
comitantly over-expression of COX-2 were also reported (Farsandaj
et al., 2012). In addition, phytocannabinoids were shown to be as
effective as synthetic compounds (Ligresti et al., 2006). Particu-
larly, CBD was reported to be effective at inhibiting cell prolifera-
tion of both hormone-sensitive (MCF-7 ) and hormone-negative
(MDA-MB-231) cells showing a combination of cell type depen-
dent mechanisms of action which include either direct or indirect
activation of CB2 and TRPV1 receptors and induction of oxidative
stress, all contributing to induce apoptosis. The efficacy of CBD was
also corroborated with in vivo data in athymic mice injected with
human MDA-MB-231 breast carcinoma cells (Ligresti et al., 2006).
Indeed, it was also shown that in MDA–MB231, a human breast
cell line, CBD induced endoplasmic reticulum stress, inhibition of
AKT/mTOR pathway, and up-regulation of autophagy-mediated
cell death (Shrivastava et al., 2011). Recent studies indicated an
anticancer activity induced by a quinone/cannabinoid derivative
through the activation of CB2 receptors and oxidative stress me-
chanism to induce apoptosis in triple-negative breast cancer, a
highly aggressive type of breast cancer (Morales et al., 2015). Other
recent studies indicated that CBD significantly inhibits epidermal
growth factor (EGF)-induced proliferation and chemotaxis of
breast cancer cells. In addition, it was shown that CBD inhibits
EGF-induced activation of EGFR, ERK, AKT and NF-kB signalling
pathways as well as MMP2 and MMP9 secretion (Elbaz et al.,
2015). CBD also inhibited tumour growth and metastasis in dif-
ferent mouse model systems. Analysis of molecular mechanisms
revealed that CBD significantly inhibits the recruitment of tumour-
associated macrophages in primary tumour stroma and secondary
lung metastases (Elbaz et al., 2015). Fig. 3 summarizes the im-
portant pathways in cannabinoid induced cytotoxicity.
4. Cannabinoids and brain cancer
Both CB1 and CB2 receptors have been identified in the CNS
(Ameri, 1999; Benito et al., 2007; Herkenham et al., 1991; Nunez
et al., 2004; Skaper et al., 1996). High density of CB1 receptors has
been reported in different areas of the brain such as in the cortex,
cerebellum and hippocampus (Herkenham et al., 1991; Hoffman
et al., 2010; Sullivan, 2000; Tsou et al., 1998). The CB1 receptor
protein is mainly localised in astroglial cells and neurones whereas
CB2 receptors are located on microglial cells (Held-Feindt et al.,
2006; Stella, 2004) with possible neuroprotective activity (Cabral
and Griffin-Thomas, 2008; Kreitzer and Stella, 2009) and in some
benign paediatric astrocyte tumours (Ellert-Miklaszewska et al.,
2007). The anti-tumour effect of cannabinoids on gliomas, glio-
blastoma multiforme or astrocytoma that are the most frequent
class of malignant primary brain tumours, will be discussed below.
Although the downstream events by which cannabinoids exert
their action are not completely elucidated, it can be generally as-
sumed that they act at least through two mechanisms: induction
of apoptosis of tumour cells and/or inhibition of tumour angio-
genesis and migration.
The anti-tumour action of two cannabinoid receptor agonists,
Δ9-THC and WIN-55,212–2, was shown to be mediated by an in-
crease in the level of ceramide leading to the activation of extra-
cellular signal-regulated kinase (ERK1/2) in C6 glioma cells (Galve-
Roperh et al., 2000; Guzmán, 2003). In addition, positive actions of
CP 55–940 include a selective induction of cell death in a hybrid
cell line of neuroblastoma plus glioma where consistent anti-
proliferative effects were observed (Tomiyama and Funada, 2011).
It was of interest to note that CP 55–940 had a selective and dif-
ferential action on C6 compared to the U373 astrocytoma cell lines.
Results showed that C6 cells were dying faster than the U373 cells
mainly via necrotic mechanisms after being exposed to CP 55–940,
whilst U373 cells underwent early apoptosis and displayed a more
defined laddering pattern (Ortega et al., 2015). This illustrates that
the effects of cannabinoids on cancer cell lines in vitro is context
specific.
Other studies (Ellert-Miklaszewska et al., 2005) also showed a
down regulation of phosphoinositide 3-kinase (PI3K), protein ki-
nase B (Akt), and ERK signalling pathways, and activated pro-
apoptotic function of Bad protein, which lead to induction of
apoptosis. In similar experiments using the CB2 receptor agonist
 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14
JWH-133, apoptosis was induced in glioma cells via enhanced
ceramide synthesis de novo (Sánchez et al., 2001a; Sarfaraz et al.,
2008). However, in addition to ceramide pathway, other pathways
such as stress-regulated protein p8 which leads to the activation of
activating transcription factor-4 (ATF-4) and cell death-inducible
kinase (TRB3) were shown as mechanism/s of the anti-tumour
action of cannabinoids (Carracedo et al., 2006a). Other studies
have demonstrated that CBD, via receptor-independent manner,
triggers apoptosis of human glioma cells by a cellular mechanism
that involves an early production of reactive oxygen species (ROS),
depletion of glutathione (GSH), and concomitant activation of
caspase cascade with no effect in non-transformed cells (Massi
et al., 2006). Moreover, the same group, reported that CBD can
inhibit proliferation and invasion of different glioma cell lines
through a multi-target mechanism affecting the most relevant
pro-tumour ERK and PI3K/Akt signalling pathways, as well as the
expression of the transcription factor HIF-1α which was down
following a treatment with CBD. Such experiments were con-
ducted under ‘pseudo-hypoxic conditions’ (Solinas et al., 2013).
This raises the possibility that these compounds could have ac-
tivity against hypoxic cells but to the best of our knowledge, no
studies of this nature have been conducted.
Remarkably, the cannabinoid-mediated anti-proliferative ac-
tion appears to be selective for brain-tumour cells as the survival
of normal brain cells astrocytes (Gomez Del Pulgar et al., 2002),
oligodendrocytes (Molina-Holgado et al., 2002) and neurons
(Mechoulam et al., 2002) are unaffected or even favoured by
cannabinoid challenge. Accordingly, a pilot phase I clinical trial, in
a cohort of recurrent glioblastoma multiforme tumour patients
expressing cannabinoid receptors reported an anti-proliferative
action for Δ9-THC on tumour cells with an acceptable safety profile
(Guzman et al., 2006). Further phase 1b/2a clinical trials are un-
derway and await publication.
Based on these findings, other studies evaluated the expression
of cannabinoid receptors in surgical material of solid astrocytomas,
gliomas and glioma cell lines. Whilst CB1 expression was slightly
increased in astrocytomas and gliomas, the CB2 expression was
similar in both tumour and normal brain tissue. The authors found,
in accordance with this receptor subtype expression in situ, that
agonists selective for CB1 or active on both subtypes reduced
elevated cyclic AMP levels and cell proliferation, but failed to in-
duce apoptosis in glioma cells in vitro (Held-Feindt et al., 2006).
Further experiments described opposite changes in CB1 and CB2
receptor protein expression in human gliomas (López-Moreno
et al., 2010). The reduction in the level of CB1 receptor expression
or mRNA in glial tumours is suggested to be related to the neu-
ronal loss (Canoll and Goldman, 2008). The reduction in CB1 re-
ceptor expression was in line with a reduction in the WIN 55,212–
2 stimulated [35S]GTPγS binding to glioblastoma multiforme
membranes. This suggested a reduction in the number of available
receptors in the glioblastomas. Similar observations were reported
in the brain of aged rats (Romero et al., 1998). It should be noted
that similar reductions were also observed in neurodegenerative
diseases such as Alzheimer, Parkinson and Huntington diseases,
and also in normal aging (Glass et al., 1993; Hurley et al., 2003;
Richfield and Herkenham, 1994; Westlake et al., 1994). In parti-
cular the up-regulation of CB2 receptors has also been reported in
other disorders such as Alzheimer, Huntington diseases, en-
cephalitis and multiple sclerosis (Benito et al., 2007, 2003; Fer-
nandez-Ruiz et al., 2007). It is suggested that reduction in the level
of CB1 receptors occurs as a result of neuronal loss, possibly si-
multaneous to the enhanced brain gliosis that appears with nor-
mal aging. In addition to an up-regulation of CB2 receptors in
malignant tumours, the level of anandamide was reported to in-
crease in glioblastomas (Petersen et al., 2005). It can then be
suggested that the endogenous anandamide will bind to CB2
7
receptors to mediate an anti-tumour activity. This is in line with
studies by Sánchez et al in 2001 who reported a considerable re-
gression of malignant glioma cells by the local administration of a
CB2 selective agonist (Sánchez et al., 2001a). In such studies ad-
ministration of JWH-133 to an immunocompromised mice model,
Rag-2  /  mice inoculated with rat glioma C6-cells caused a 71%
reduction in the tumour growth as compared to the control group
and was found to be prevented by co-administration of the CB2
receptor antagonist, SR144528 but not the CB1 receptor antagonist,
SR141716. Further experiments by the same group indicated that
the anti-tumour effects induced by the activation of CB2 receptors
by JWH-133 initiated apoptosis via ceramide synthesis and ERK1/2
activation (Sánchez et al., 2001a). In later studies by Blazquez and
colleagues in 2003 using the same mouse model, intra-tumour
administration of JWH-133 showed a significant reduction in
mRNA expression of the pro-angiogenic factors, vascular en-
dothelial growth factor and angiopoietin 2 (Blazquez et al., 2003).
Additional data to support the importance of CB2 receptors in
glioblastoma came from studies that showed an increase in the
level of CB2 receptors in the endothelial cells of human glio-
blastoma vessels (Schley et al., 2009). Whilst the mechanism of
action of CB agonists against glioblastoma is still not fully under-
stood, it has been shown in the rat C6 glioma cells that the high
affinity glycine transporter (GLYT1) has been attenuated via pro-
tein kinase C alpha (PKC-α) (Morioka et al., 2008). Attenuation of
GLYT1 would increase the inhibitory action of glycine transmitter
in the synapse. Other studies showed that PKC inhibitors could
impair the CB effects in neuroblastoma cells (Rubovitch et al.,
2004). The above data suggest the involvement of downstream
PKC and GLYT1 regulation in mediating an anti-glioblastoma ac-
tivity by cannabinoids. Thus, the accumulated data suggest that
the anti-tumour activity of cannabinoids could be mediated via
ERK1/2, AKt and/or PI3K pathways as well as PKC and glycine
transporters. The change in the levels of cannabinoid receptor
expression and the levels of endocannabinoids depend on the
stages of cancer. For example, the levels of CB2 receptors and
anandamide increase in advanced tumours.
5. Cannabinoid and lung cancer
The first evidence of the antineoplastic activity of cannabinoids
against lung cancer dates back to 1975 when Munson et al de-
monstrated a dose-dependent retardation in tumour growth in the
Lewis lung adenocarcinoma animal model (Munson et al., 1975).
Later on, further studies were carried out in order to elucidate the
possible mechanism of action(s) of this class of molecule although
controversial evidence about the anti-tumour action of cannabi-
noids were reported for this particular type of cancer. In fact, it has
been reported that Δ9-THC suppresses the host immune reactivity
against lung cancer and that the augmentation of tumour growth
acts through inhibition of anti-tumour immunity by a CB2 re-
ceptor-mediated, cytokine-dependent pathway (Zhu et al., 2000).
Δ9-THC, although without the modulation of EGFR expression or
FAK phosphorylation previously reported by others (Hart et al.,
2004), has been also shown to attenuate the EGF-induced migra-
tion and invasion of epidermal growth factor receptor-over-
expressing lung cancers, which are often highly aggressive and
resistant to chemotherapy (Preet et al., 2008). Furthermore, in
in vivo experiments, administration of Δ9-THC suppressed metas-
tasis and subcutaneous tumour growth in severe combined im-
munodeficient mice (Preet et al., 2008). The anti-invasive effect of
cannabinoids has also been reported (Ramer and Hinz, 2008; Ra-
mer et al., 2010). In these studies, a cannabinoid receptor and
TRPV1-triggered expression of tissue inhibitor of matrix metallo-
proteinases-1 (TIMP-1) was identified as an important mediator of
8 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14
the anti-invasive action of cannabidiol. Impaired invasion driven
by CBD links the cannabinoid and TRPV1 receptors to the activa-
tion of MAPK pathways and subsequent TIMP-1 induction. Ad-
ditionally and in line with its in vitro anti-invasive action, in vivo
studies in thymic-aplastic nude mice revealed a significant in-
hibition of A549 lung metastasis in cannabidiol-treated animals as
compared to vehicle-treated controls (Ramer and Hinz, 2008;
Ramer et al., 2010).
The expression of CB1 and CB2 receptors has been reported in
non-small cell lung cancer (NSCLC) patients and also the NSCLC
cell lines, A549 and SW-1573 (Preet et al., 2011). Pre-treatment of
A549 and SW-1573 cells with WIN 55,212–2 and JWH-015 reduced
chemotaxis and chemo-invasion as well as migration and these
were sensitive to blockade by the CB1 and CB2 receptor antago-
nists, AM251 and AM630 respectively. Furthermore both agonists
revealed a reduction in tumour growth in vitro, inhibited in vivo
tumour growth and lung metastasis that again were sensitive to
antagonism of CB1 and CB2 receptors (Preet et al., 2011). Me-
chanistic studies revealed an inhibition of phosphorylation of Akt
and MMP-9 expression/activity upon exposure to the CB1 and CB2
receptor agonists in NSCLC (Preet et al., 2011). On the other hand,
the involvement of cannabinoid receptor is not the only me-
chanism through which cannabinoids exhibit their anti-tumour
proprieties. In fact, as demonstrated by Gardner et al. these mo-
lecules can exert some of their biological effects via modulation of
prostaglandin production. This study has shown that administra-
tion of methanandamide at 5.0 mg kg  1 in murine lung cancer
could increase the rate of tumour growth (both in vitro and
in vivo). Such studies also showed an increase in the level of
prostaglandin (PG) E2 and COX-2 which were sensitive to blockade
by the COX-2-specific inhibitor, SC58236, the p38/MAPK inhibitor,
SB203528, and the p42/44 inhibitor, PD98059 but not to CB1 and
CB2 receptor antagonists. The results confirmed an up-regulation
of COX-2 which was independent of cannabinoid receptor activa-
tion (Gardner et al., 2003). Recently studies have shown an up-
regulation of COX-2 induced by CBD in two different lung cancer
cell lines (A549, H460) and primary cells from a patient with lung
cancer (Ramer et al., 2013). Such studies showed a pro-apoptotic
and tumour-regressive action by CBD through an initial up-reg-
ulation of COX-2 and PPAR-γ and a subsequent nuclear transloca-
tion of PPAR-γ by COX-2-dependent prostaglandins, PGD2 (Ramer
et al., 2013). Such studies also indicated that incubation of the cells
with PGD2 but not PGE2 was associated with a concentration-
dependent loss of cell viability. This highlighted how different
cannabinoids could induce differential effects, which are CB1 and
CB2 receptor-independent pathways and also dependent on the
type of PG being produced.
In further recent studies, it was demonstrated that cannabi-
noids induced up-regulation of intercellular adhesion molecule 1
(ICAM-1) on lung cancer cells to be responsible for increased
cancer cell lysis by lymphokine-activated killer (LAK) cells (Haus-
tein et al., 2014). This recent study suggested a new mechanism for
anti-tumour activity of cannabinoids. Further experiments by Ra-
mer et al also indicated that cannabinoids induce anti-angiogenic
effect in endothelial cells via the release of TIMP-1 from lung
cancer cells (Ramer et al., 2014).
Thus, the likely pathways involved in mediating anti-tumour
activity induced by cannabinoids are CB1, CB2 and TRPV1 receptors
as well as MAPK and TIMP-1 pathways. However, this does not
negate a role for PPAR-γ and COX-2-dependent prostaglandins.
6. Cannabinoids and intestinal cancer
Endocannabinoid signalling has been proved crucial for certain
aspects of gastrointestinal homoeostasis. It also plays an essential
role in the regulation of intestinal tumour growth. Recent studies
have demonstrated an up-regulation of anandamide and its me-
tabolite arachidonic acid in cancer tissues of patients with colon
cancer with lymphatic metastasis (Chen et al., 2015). In addition,
CB1 receptor expression was elevated (Chen et al., 2015). Another
recent study also demonstrated that the level of expression of CB2
receptors correlates with cancer progression and can predict pa-
tient survival in colon cancer patients. Such studies showed that
high levels of CB2 receptors correlate with poor prognosis in pa-
tients with tumours in advanced stages or with vascular invasion
(Martinez-Martinez et al., 2015).
There are however several controversies regarding the role of
the cannabinoid receptors in colorectal cancer (Izzo and Camilleri,
2009). In adenomatous polyposis coli (APC) gene knock-out
models (mutation of the gene leads to colon cancer), mice with an
additional deletion in the cannabinoid receptor 1 (CNR1) gene or
subjected to pharmacological blockade of the CB1 receptor, de-
monstrated a higher colonic tumour burden than their littermates
whereas activation of CB1 attenuated intestinal tumour growth by
inducing cell death via down-regulation of the anti-apoptotic
factor survivin (Wang et al., 2008). In contrast to these findings,
the CB1 antagonist rimonabant inhibited the growth of cancer cells
and the development of precancerous lesions in mice (Santoro
et al., 2009). Other studies showed that non-selective cannabinoid
receptor agonists such as anandamide, 2-AG and HU-210, and an
inhibitor of anandamide inactivation, potently inhibited human
epithelial colorectal adenocarcinoma cells (CaCo-2 cell) prolifera-
tion (Ligresti et al., 2003). This effect was less prominent in a less
aggressive human colon carcinoma cell line (DLD-1 cells). The cell
proliferation effect afforded by HU-210 was inhibited by the CB1
and CB2 receptor antagonists, rimonabant and SR144528 respec-
tively, only in DLD-1 cells and not in CaCo-2 cells. This suggested
the involvement of both CB1 and CB2 receptors in mediating an
inhibition of cell growth (Ligresti et al., 2003). The differential
effects observed in the two different cell lines might be due to
differences in the level of cannabinoid receptor expression. Indeed,
it was noted that CaCo-2 cells express CB1 receptors but not CB2
receptors and DLD-1 cells express both CB1 and CB2 receptors,
with CB1 receptor less expressed than in CaCo-2 cells. Later studies
suggested that cells with high expression of cyclooxygenase-2
(COX-2) might be a target for the inhibitory action of anandamide
on cell death in colorectal carcinoma cells (Patsos et al., 2005).
Indeed CB2 receptor expression has been reported in human
adenomatous polyps and carcinomas and in human colonic epi-
thelial cell lines (Greenhough et al., 2007; Ihenetu et al., 2003;
Ligresti et al., 2003; Wright et al., 2005). Interestingly the normal
epithelial cells do not express CB2 receptors (Wright et al., 2005).
This suggests that the CB2 receptors are inducible in inflamed
tissues or tumour cells. This suggestion is in line with other studies
which reported an increase in the level of CB2 receptor expression
associated with increased differentiation, proliferation, disease
and malignancy (Fernandez-Ruiz et al., 2007; Mallat and Lo-
tersztajn, 2008). Ceramide synthesis following an increase in the
level of tumour necrosis factor (TNF)-α and activation of epidermal
growth factor receptor have been identified as the possible mo-
lecular mechanism following the activation of CB2 receptors in
colon cancer (Cianchi et al., 2008; Hart et al., 2004). Thus since
activation of CB2 receptors seem to be beneficial in cancer therapy,
further studies are needed to investigate the dual role for CB2
receptors in intestinal regeneration and anti-tumour activity. It is
also possible that different subtypes of CB2 receptors mediate
different roles, however no evidence has yet been reported.
Cannabinoids have been also reported to exert chemopreven-
tive effects in an experimental model of colon cancer, an effect
associated with down-regulation of phospho-Akt and up-regula-
tion of caspase-3 (Aviello et al., 2012). In vitro studies by the same
 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14
group on colorectal carcinoma cells demonstrated that anti-pro-
liferative effects were exerted through multiple mechanisms, in-
cluding involvement of CB1 receptors, TRPV1 and PPAR-γ (Aviello
et al., 2012). Moreover, studies by Notarnicola and collaborators
firstly described the up-regulation of CB1 expression by 17β-es-
tradiol as a further mechanism by which estrogens control colon
cancer cell proliferation (Notarnicola et al., 2008). Proto and col-
leagues confirmed these findings and reported an interaction be-
tween the endocannabinoid system and steroid hormones in the
growth of colon cancer cells. Such studies revealed that both
anandamide and 17β-estradiol inhibited proliferation of human
colorectal cancer cell lines, SW620 and DLD-1 via interaction with
CB1 receptors. Both agonists increased the CB1 receptor expression
in both cell lines by acting at the same CNR1 gene. Interestingly
the up-regulation of CB1 receptors induced by anandamide ana-
logueue was through PPAR-γ and RXRα, (Proto et al., 2012). The
data suggested that CB1 receptor is a target for 17β-estradiol and
that the endocannabinoid system could present a tool to improve
treatment in patients with colorectal cancer.
7. Cannabinoids and reproductive system cancer
During the last decade, increasing evidence has pointed to-
wards the relevance of endocannabinoids in both female and male
fertility. This association has been supported by the tightly
modulated expression of cannabinoid receptor found in gonadal
tissues. Along the male reproductive tract, CB receptors have been
detected in the testis, Sertoli cells, prostate and vas deferens (Gye
et al., 2005; Maccarrone et al., 2003; Pertwee et al., 2002; Rossato
et al., 2005). CB receptors have also been found in various parts of
the mammalian female reproductive system. In the mouse re-
productive tract, CB receptors were expressed in the uterus, ovi-
duct and also in pre-implantation embryos (Das et al., 1995; Paria
et al., 2001; Wang et al., 2004). Moreover, this localisation has also
been described in the human uterus (Dennedy et al., 2004; Iuvone
et al., 2008) and placenta during pregnancy (Habayeb et al., 2008;
Helliwell et al., 2004; Park et al., 2003).
The influence of cannabinoids on the proliferation of human
cervical adenocarcinoma cells and on macromolecular biosyn-
thetic events associated with the proliferative process were re-
ported in the late 1970s when cannabinoids induced growth in-
hibition of HeLa cells, human cervical cancer cell lines (Blevins
et al., 1980; Mon et al., 1978). More recently, an up-regulation of
both cannabinoid receptors in human ovarian cancer cells OVCAR-
3 and SKOV-3 compared to normal Chinese hamster ovarian (CHO)
cells was found. These findings led to the suggestion that these are
targets for new therapies for ovarian cancer (Afaq et al., 2006).
Recently studies also showed that CB1 receptor levels are also in-
creased and correlate with disease severity in human epithelial
ovarian tumours and this has been proposed to be an important
factor of bad prognosis following surgery in stage IV colorectal
cancer (Messalli et al., 2014; Jung et al., 2013). WIN-55,212–2 was
shown to exert, via CBR dependent manner, a decrease in cell
viability, G1 arrest in cell cycle progression, induction of apoptosis
and down-regulation of the expression of PCNA and VEGF (Afaq
et al., 2006). An abnormal expression of CB2 receptor has also been
reported in biopsies of women affected by endometrial carcinoma.
Interestingly, the up-regulation was only found in transformed
malignant cells and the staining of CB2 was completely absent in
the normal endometrial tissue from the same biopsy (Guida et al.,
2010). These findings, together with previous evidence that the
endocannabinoid system controls cell survival/death decisions
(Guzman et al., 2002) by inhibiting or stimulating cell growth,
suggest that CB2 receptors might play an important role in the
growth of endometrial carcinoma. The study revealed that the
9
complete endogenous machinery for CB2 activation was altered in
endometrial adenocarcinoma, because the levels of 2-AG, the most
efficacious endogenous CB2 agonist, were elevated, possibly as a
result of the decrease in the expression of monoglycerol lipase
(MAGL) an important enzyme necessary for 2-AG breakdown. On
the other hand, CB1 receptors and AEA, a more selective en-
dogenous agonist for CB1, as well as FAAH, the most important
AEA-metabolising enzyme, although expressed in healthy en-
dometrial tissues, remained unchanged after cell transformation
(Guida et al., 2010).
Additional proteins other than cannabinoid receptors have
been considered as possible targets in reproductive cancers. Mul-
tidrug resistance-related protein 1 (MRP1) or ATP-binding cassette
(ABC) transporter, ABCC1, is a membrane-bound, ubiquitously
expressed energy-dependent efflux transporter. In terms of phy-
siological function, it is involved in transporting a range of glu-
tathione, glucuronide, sulphate conjugates and cancer drugs, in-
cluding folate based anti-metabolites, anthracyclines, plant-de-
rived vinca alkaloids and anti-androgens (Cole et al., 1994; Flens
et al., 1996; Hooijberg et al., 1999; Keppler et al., 1997). Whilst the
transportation of metabolites by ABCC1 leads to attenuation of the
toxicity of such metabolites might be beneficial, efflux of cancer
drugs would however reduce intracellular concentrations in tu-
mour cells and hence induce drug resistance (Karászi et al., 2001;
Norris et al., 1996; Wijnholds et al., 2000, 1998). It has been shown
that phytocannabinoids are modulators of the ABC transporters,
ABCG2 and P-glycoprotein (Holland et al., 2007; Zhu et al., 2000).
It was also shown in the human ovarian carcinoma cell line that
cannabinoids such as cannabinol, cannabidiol and Δ9-THC in-
creased the intracellular accumulation of two ABCC1 substrates,
Fluo3 and the cancer drug, vincristine, in 2008/MRP1 cells (the
human ABCC1 transduced subline) (Holland et al., 2008). In such
experiments cannabidiol was shown to be the most potent and Δ9-
THC to be the least potent cannabinoid. The rank order of potency
for ABCC1 inhibition was independent of the substrate assayed
(Holland et al., 2008). Further pre-clinical studies are required to
establish if inhibition of ABC transporters by cannabinoids can
alter the disposition and efficacy of therapeutic drugs that are
substrates for these transporters.
With regard to the male reproductive system and its physiol-
ogy, the antagonising effect of cannabinoids can be dated back to
1974 where experimental models in male rats showed depression
of spermatogenesis (Dixit et al., 1974) and decrease in circulating
testosterone levels (Kolodny et al., 1974). Endocannabinoids,
through interaction with CB1 receptors and synthetic en-
docannabinoid-vanilloid hybrids via stimulation of TRPV1 chan-
nels have been shown to inhibit nerve growth factor (NGF)-in-
duced proliferation of human prostate PC-3 cells (Melck et al.,
2000). However, THC was suggested to induce apoptosis of these
cells via a receptor-independent mechanism (Ruiz et al., 1999), but
also increase the production of the pro-proliferative factor, NGF
(Velasco et al., 2001). Later studies showed an increased expres-
sion of both CB1 and CB2 receptors in cultured prostate cancer cells
when compared with normal prostate cells. Moreover, treatment
of prostate cancer cells with WIN-55,212–2 resulted in a dose and
time dependent decrease in cell viability, increased apoptosis
along with decrease in androgen receptor protein expression, PSA
expression, and secreted PSA, suggesting that cannabinoids should
be considered as agents for the management of prostate cancer
(Ruiz-Llorente et al., 2003; Sanchez et al., 2003; Sarfaraz et al.,
2005). It was also found that a high level of CB1 receptor im-
munoreactivity (CB1IR) in prostate cancer tissues is associated
with the severity and outcome of the disease (Chung et al., 2009).
It was then suggested that the effect of cannabinoids on pros-
tate cancer cells depends on the concentration of cannabinoids
used and the incubation time. Cannabinoids used at
10 F.A. Javid et al. / European Journal of Pharmacology 775 (2016) 1–14
concentrations lower than micromolar induced androgen receptor
expression whilst at higher concentrations induced apoptosis or
cell-cycle arrest (Mimeault et al., 2003; Sanchez et al., 2003; Sar-
faraz et al., 2006). It was also shown that CB1 receptor antagonists
prevented/blocked the anti-tumour activity of agonists whilst a
longer incubation time failed to reveal any antagonist effect (Mi-
meault et al., 2003; Sarfaraz et al., 2005).
Other studies have also indicated an important role for CB2 but
not CB1 receptors in the anti-proliferative effect of cannabinoids
(Olea-Herrero et al., 2009). Such studies have shown that in PC-3
cells pre-treated with rimonabant failed to reduce the effect of
methanandamide on cell cycle and apoptosis. However, pre-
treatment with the CB2 receptor antagonist, SR 144528 attenuated
the number of apoptotic cells and the number of sub-G1 cells in-
duced by methanandamide and apoptosis afforded by the CB2
receptor agonist, JWH-015. Furthermore, when CB2 receptor ex-
pression was significantly reduced by siRNA, apoptosis afforded by
JWH-015 was completely antagonised thus further indicating an
involvement of CB2 receptors in apoptosis (Olea-Herrero et al.,
2009).
Recent studies have focused on the role of non-selective, cal-
cium permeable cation channels of the transient receptor potential
(TRP) channels in prostate cancer initiation and progression.
Capsaicin, a natural ligand for TRPV1, has been reported to elicit
both pro-proliferative and pro-apoptotic effects on prostate cancer
cell lines (Czifra et al., 2009; Malagarie-Cazenave et al., 2009, 2011;
Sánchez et al., 2006, 2005; Ziglioli et al., 2009). Moreover, it has
been suggested that TRP channels of melastatin-type 8 (TRPM8)
are over-expressed in androgen-dependent prostate cancer cell
lines in a manner dependent on androgen receptor (AR) activation
(Bidaux et al., 2007, 2005; Henshall et al., 2003; Tsavaler et al.,
2001; Zhang and Barritt, 2004). Several studies have shown that
cannabinoids antagonise TRPM8 channels and activate and sub-
sequently desensitise TRPV2 and TRPV1 channels (De Petrocellis
et al., 2011, 2008; Qin et al., 2008). Further recent studies showed
an inhibition of cell viability and induction of apoptosis by can-
nabinoids when tested in serum protein-deprived medium sug-
gesting an intracellular target for the cannabionids (De Petrocellis
et al., 2013). However, the molecular mechanism of action was
demonstrated to be due not uniquely to a direct TRPM8 antag-
onism, but rather to AR down regulation, which in turn can lead to
TRPM8 down-regulation. As it has been suggested that estrogens
are involved in the survival of prostate cells, the authors also ex-
amined the involvement of those receptors and they found that
GPR30, rather than estrogen metabolic enzymes or ERα or ERβ,
may be one of the intracellular targets through which cannabi-
noids stimulate the ER branch of the intrinsic pro-apoptotic
pathway Δ9-THC (De Petrocellis et al., 2013).
In addition, the expression of a potential cannabinoid receptor,
GPR55 has been also shown at the mRNA and protein level in both
ovarian and prostate cancer cell lines (Pineiro et al., 2011). Al-
though the physiological role of this receptor is not fully under-
stood, it is suggested to have important roles in regulating pro-
liferation and growth (Pineiro et al., 2011). This novel receptor is
suggested to be associated with lysophosphatidylinositol (LPI) to
induce calcium mobilisation and activation of Akt and extracellular
signal-regulated kinase (ERK)1/2 in ovarian and prostate cell lines.
Blockade of this receptor might have novel therapeutic potential in
managing ovarian and prostate cancer. All these findings indicate
that cannabinoids can retard proliferation and cause apoptosis of
PCC via a combination of cellular and molecular mechanisms
(cannabinoid receptor-mediated and/or independent). These stu-
dies are encouraging and they support the development of clinical
studies on these molecules as a therapy for human prostate car-
cinoma, either as single agent or in combination with existing
compounds.
8. Conclusion and future direction
The substantial knowledge of palliative and anti-tumour ac-
tions of cannabinoids gained by the scientific community in the
last few years has raised the profile of these molecules and many
are promising candidates for cancer treatment. However, the use
of cannabinoids in medicine is limited by their psychoactive ef-
fects, thus cannabinoid-based therapies that are devoid of un-
wanted side effects or with a safe profile/pharmacological window
are required. A further aspect which complicates the practise of
cannabinoid-based therapies is the lack of detailed understanding
of their mechanisms of action. There is plenty of evidence in lit-
erature, mostly of them reported in this review, about the ability of
cannabinoids to induce different pathways of cell death depending
on the neoplastic cell type under investigation. On the other hand,
the effect of these compounds on several distinct hallmarks of
cancer rather than on one single process is potentially desirable.
Even though the resolution of the conflicting evidence around
cannabinoid action still remains a high research priority, some key
points need to be emphasised. Despite a small number of reports
that state their inefficacy, the vast majority of independent pre-
clinical studies report a sustained anti-tumour activity for canna-
binoids. Moreover, an important feature of cannabinoid pharma-
cology that can have important clinical implications is the lack of
toxicity frequently reported on non-tumour cells. Cannabinoid-
based medicines have been already proven to be safe in thousands
of patients enroled in clinical trials for cancer patients (Gro-
tenhermen, 2007; Portenoy et al., 2012; Robson, 2011). This
highlights a need for the identification of the molecular mechan-
isms which confer sensitivity to this class of drugs. Another ap-
pealing possibility consists of adding to a standard therapy a
mixture of molecules able to directly or indirectly target the en-
docannabinoids system in order to enhance anti-tumour actions of
chemotherapy and attenuate unwanted iatrogenic side effects.
Indeed it is interesting to note that the administration of THC and
CBD enhanced the radio-therapeutic effect in an orthotopic mur-
ine glioma model (Scott et al., 2014). It is proposed that CBD could
alleviate the THC-induced side effects such as convulsion, dis-co-
ordination, and psychotic episodes, thus it was suggested that the
administration of CBD in combination with THC may help to im-
prove the tolerability to cannabinoids (Pertwee, 2009). Finally,
only the improvement of basic research will lead to the identifi-
cation of the most appropriate patient population for a cannabi-
noid-based therapy and will facilitate the acceptance of cannabi-
noid use in the clinic.
Conflict of interests/acknowledgement
FJ and AL disclose that they have received research support
funding from GW Pharma.
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